Cardiac myosin is the most abundant and bioactive protein presented in heart tissue. In this work, a simple method was described for preparation of high purity cardiac myosin for further research. With the use of a domestic pig left ventricle as the starting tissue, this method yielded 4.5 mg myosin per gram of wet tissue with a specific activity of 0.293 U/mg. The purification factor was 4.9-fold with an activity recovery of 42.1%. In addition, the investigation of fluorescence spectrum shown that the emission intensity of cardiac myosin has a dramatic decrease under the influence of an acidic environment or at temperatures higher than 30 °C, which could influence the normal physiological function of cardiac myosin.